In-vitro diagnostic tests via immunoassays typically involve antibodies with specific binding affinity toward the analytes of interest. In such test configurations, antigenic analytes either bind directly with the antibody or compete with a hapten-label conjugate (competition assay).
In the former case, the antibody-antigen complex may be allowed to grow as in agglutination assays. Alternatively, in sandwich assays, another antibody with affinity to the antigen can be conjugated to a signal label and allowed to bind the primary antibody-antigen complex. The signal response is directly proportional to the analyte concentration.
In competition assays, competition is established between the analytes, typically small molecules in nature, and the hapten-label conjugates. The signal response is inversely proportional to the analyte concentration.
Most target analytes can, in principle, be detected using either the competition assay or the sandwich assay format. While there are numerous examples where the stated methods served the purpose very well, the utility of these approaches however, were somewhat limited in that a single assay is specific for a single analyte only. A multi-analyte assay is difficult.
The following exemplifies the different available immunoassay formats. Mochida et al., U.S. Pat. No. 4,185,084 discloses non-homogeneous assays with wash and separation steps. An insolubilized anti-analyte antibody acts as a primary capture phase for the analyte which is conjugated to a hapten (hapten-analyte conjugate). After washing, soluble labelled anti-hapten antibody is added and the complex of (anti-analyte antibody/hapten-analyte conjugate/anti-hapten antibody) is detected. Sadeh et al, U.S. Pat. No. 4,243,749, discloses another similar sandwich assay format. Sadeh et al., is specifically oriented towards measuring low molecular weight (hapten) antigens. The unknown analyte and the hapten-analyte conjugate are incubated together with the insoluble anti-analyte antibody in a competitive assay format. Following the wash step, soluble labeled anti-hapten antibody is added and the system is washed again and the labeled complex of (anti-analyte antibody/hapten-analyte conjugate/anti-hapten antibody) detected.
Kang et al., Clin. Chem., 32(9):1682-1686 (1986) describes two assay formats. The first assay format presents an enzyme immunoassay which uses an anti-hapten antibody coated microparticle (the common capture particle), an anti-analyte antibody conjugated to a hapten (hapten-anti-analyte-antibody conjugate), and a labeled anti-analyte antibody. If the analyte is present in a sample, a complex of (anti-hapten antibody coated microparticle/hapten-anti-analyte-antibody conjugate/analyte/labeled anti-analyte antibody) is formed and detected. Fluorescein serves as the capture hapten. The microparticle is a latex particle.
Kang et al.'s second assay format is similar to that of Bunting, U.S. Pat. No. 4,271,140. The assay format consists of an anti-hapten antibody bound to a solid phase, a hapten conjugated to an anti-analyte antibody (hapten-anti-analyte antibody) and labeled analyte. The complex of all three components are detected.
In agglutination assays, either the antibodies or the antigens (or hapten) may be bound to small particles. The particles that have been used as agglutinable carriers include latex, charcoal, kaolinite, bentonite, inorganic colloidal particles, as well as both microbial cells and erythrocytes. See Mochida, U.S. Pat. No. 4,308,026. When these coated particles (coated with either antibodies or antigens) are mixed with samples containing antigens or antibodies, the coated particles would form visually detectable agglutination. Agglutination is characterized by the clumping of the latex polymer particles from an otherwise smooth suspension. Qualitative latex agglutination tests can be carried out on a simple slide without the aid of any instrumentation. More than one antigen can be detected simultaneously by means of differently colored latex particles which have each been sensitized with antibodies of different specificity and then mixed together. Hadfield, S. G., et al., J. Immunol Methods, 97: 153-8 (1987). Hadfield et al, U.S. Pat. No. 4,745,075 May 17, 1988. U.S. Pat. No. 4,419,453 further discloses latex particles dyed with Amocid yellow, brilliant crocein 3BA red dye, and Calco Oil Blue N Dye. U.S. Pat. No. 4,745,075 discloses that there are also marketed test kits for the grouping of Beta Haemolytic Streptococci which include reagents in which the solid phase is a suspension of killed red-dyed or blue-dyed Staphylococcus aureus cells.
Hillyard et al., U.S. Pat. No. 5,086,002, discloses an erythrocyte agglutination assay in which the agglutination reagent comprises at least one erythrocyte binding molecule coupled to at least one specific analyte binding molecule wherein the erythrocyte binding molecule does not cause agglutination when incubated with erythrocytes in the absence of analyte (in the case of a direct assay) or analyte binding reagent (in the case of an indirect assay). The erythrocytes are preferably endogenous to the blood sample to be tested. Mixtures of conjugates and conjugates of analyte analogues with erythrocyte binding molecules may also be used as agglutination reagents. Chang, U.S. Pat. No. 4,433,059, discloses an agglutination immunoassay reagent in which two antibodies are covalently linked "tail-to-tail", so as not to alter their specificity. One antibody is specific for an antigen borne by an indicator substance, such as an erythrocytes.